The invention is directed to anti-atherosclerotic agents and more specifically, to compounds, compositions and methods for treating atherosclerotic conditions, such as dyslipoproteinemias and coronary heart disease. This invention specifically relates to amino thioxomethyl amino oxyacetic acid derivatives that elevate HDL cholesterol concentration and which may be useful for the treatment of atherosclerotic conditions such as, coronary heart disease.
Numerous studies have demonstrated that both the risk of coronary heart disease (CHD) in humans and the severity of experimental atherosclerosis in animals are inversely correlated with serum HDL cholesterol (HDL-C) concentrations (Ross et al, Am. J. Med., 11 (1951) 480-493; Gofman et al, Circulation, 34 (1966) 679-697; Miller and Miller, Lancet 1 (1975) 16-19; Gordon et al, Circulation 79 (1989) 8-15; Stampfer et al, N. Engl. J. Med., 325 (1991) 373-381; Badimon et al, Lab. Invest., 60 (1989) 455-461). Atherosclerosis is the process of accumulation of cholesterol within the arterial wall which results in the occlusion, or stenosis, of coronary and cerebral arterial vessels and subsequent myocardial infarction and stroke. Angiographical studies have shown that elevated level of some HDL particles in humans appears to be correlated to a decreased number of sites of stenosis in the coronary arteries of humans (Miller et al, Br. Med. J., 282 (1981) 1741-1744).
There are several mechanisms by which HDL may protect against the progression of atherosclerosis. Studies in vitro have shown that HDL is capable of removing cholesterol from cells (Picardo et al, Arteriosclerosis, 6 (1986) 434-441). Data of this nature suggest that one antiatherogenic property of HDL may lie in its ability to deplete tissues of excess free cholesterol and eventually lead to the delivery of this cholesterol to the liver (Glomset, J. Lipid Res., 9 (1968) 155-167). This has been supported by experiments showing efficient transfer of cholesterol from HDL to the liver (Glass et al, J. Biol. Chem., 258 (1983) 7161-7167; MacKinnon et al, J. Biol. Chem., 261 (1986) 2548-2552). In addition, HDL may serve as a reservoir in the circulation for apoproteins necessary for the rapid metabolism of triglyceride-rich lipoproteins (Grow and Fried, J. Biol. Chem., 253 (1978) 1834-1841; Lagocki and Scanu, J. Biol. Chem., 255 (1980) 3701-3706; Schaefer et al, J. Lipid Res., 23 (1982) 1259-1273). Accordingly, agents which increase HDL cholesterol concentrations are useful as anti-atherosclerotic agents, particularly in the treatment of dyslipoproteinemias and coronary heart disease.
Ureas, thiorureas and derivatives thereof are known to be useful for treating various conditions. For example, European Patent No. 528 148 A1 discloses the use of N-phenyl thiourea derivatives (1) for the treatment of atherosclerosis by elevation of HDL-C serum levels. Among the compounds disclosed is 5-chloro-2-methylphenyl thioureido acetic acid (2). In all cases, one of R1 or R2 is hydrogen. 
U.S. Pat. No. 3,282,987 claims the use of xcex1-ureidooxy carboxylic acids (3) and their derivatives as plant growth regulators and herbicides. 
where R is hydrogen, alkyl, or chlorophenyl; and R1 is hydrogen, or alkyl of 1-12 carbons.
U.S. Pat. No. 3,625,968 discloses compounds of the formula, R1 and R2 may be identical or different and each denotes aliphatic, araliphatic, cycloaliphatic, or aromatic; R1 may also be hydrogen.
U.S. Pat. No. 3,438,985 discloses similar compounds where R1 and R2 represents hydrogen or lower alkyl.
French Patent No. 1,432,738 also discloses similar compounds where R1 is lower alkyl and R2 is hydrogen.
In accordance with this invention there is provided thioxomethyl aminoxy acetic acid derivatives of Formula I: 
wherein
R is lower alkyl;
R1 is hydroxy, amino, or lower alkoxy;
R2 and R3 are each independently hydrogen, alkyl or aryl;
Ar is phenyl, indanyl, benzhydryl, or phenyl substituted with one or more members of the group consisting of halogen, lower alkyl, perfluoroalkyl, lower alkoxy, perfluoroalkylalkoxy, dialkylamino, and aryloxy; or pharmaceutically acceptable salts thereof.
This invention also provides methods of elevating HDL concentration and treating or inhibiting atherosclerosis and related coronary heart disease or dyslipoproteinemias and increasing the HDL cholesterol concentration in a mammal in need thereof comprising administering to and said mammal an effective amount of a compound of Formula I: 
wherein
R is lower alkyl;
R1 is hydroxy, amino, or lower alkoxy,
R2 and R3 are each independently hydrogen, alkyl or aryl;
Ar is phenyl, indanyl, benzhydryl, or phenyl substituted with one or more members of the group consisting of halogen, lower alkyl, perfluoroalkyl, lower alkoxy, perfluoroalkylalkoxy, dialkylamino, and aryloxy; or pharmaceutically acceptable salts thereof.
Preferably, the present compounds are those represented by Formula I: 
wherein
R is lower alkyl;
R1 is lower alkoxy;
R2 and R3 are hydrogen;
Ar is phenyl, indanyl, or phenyl substituted with one or more members selected from the group consisting of halogen, lower alkyl, lower alkoxy, and aryloxy; or pharmaceutically acceptable salts thereof.
As used in the present application, the term xe2x80x9clower alkylxe2x80x9d and xe2x80x9clower alkoxyxe2x80x9d include both straight chain and branched moieties having 1-6 carbon atoms. The term xe2x80x9calkylxe2x80x9d also included branched and straight chain moieties and is not limited in carbon number. The term xe2x80x9carylxe2x80x9d includes aromatic radicals of 6-12 carbon atoms. The term xe2x80x9chalogenxe2x80x9d includes fluorine, chlorine, bromine and iodine.
The pharmaceutically acceptable salts of the present compounds include those derived from organic and inorganic acids such as, but not limited to: acetic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, hydrochloric, hydrotoronic, phosphoric, nitric, sulfuric, methanesulfonic, toluene sulfonic, and similarly known, acceptable acids. The most preferred compounds of this invention are:
Ethyl [[[[(5-Chloro-2-methylphenyl)amino]thioxomethyl]methylamino]oxy]acetate;
Ethyl [[[[(4-Chloro-2-methylphenyl)amino]thioxomethyl]methylamino]oxy]acetate;
Ethyl [[[[(2,3-dihydro-1H-inden-5-yl)amino]thioxomethyl]methylamino]oxy]acetate;
Ethyl [[methyl[[(2-(1-methylethyl)phenyl]amino]thioxomethyl]methylamino]oxy]acetate;
Ethyl [[[[(2,5-dimethylphenyl)amino]thioxomethyl]methylamino]oxy]acetate;
Ethyl [[[[(2-methylpropyl)amino]thioxomethyl]methylamino]oxy]acetate;
Ethyl [[[[(4-phenoxyphenyl)amino]thioxomethyl]methylamino]oxy]acetate;
Ethyl [[[[(4-Chloro-2-methylphenyl)amino]thioxomethyl]ethylamino]oxy]acetate.;
Ethyl [[[[(5-Chloro-2-methylphenyl)amino]thioxomethyl](1-methylethyl)amino]oxy]acetate; and
Ethyl [[[[(5-Chloro-2-methylphenyl)amino]thioxomethyl](1-methylpropyl)amino]oxy]acetate
The compounds of the invention may be prepared readily according to the following reaction scheme or modification thereof using readily available starting materials, reagents and conventional synthetic procedures. It is also possible to make use of variants of these process steps, which in themselves are known to and well within the preparatory skill of the medicinal chemist.
In reaction scheme IR is alkyl, R1 is alkyl, alkenyl, alkynyl, benzyl, aryl or substituted aryl, R2 and R3 are independently hydrogen, alkyl, alkenyl, alkynyl, benzyl, aryl or substituted aryl, Ar is phenyl, indanyl, benzhydryl, or phenyl optionally substituted with one or more groups selected from halogen, lower alkyl, perfluoroalkyl, lower alkoxy, perfluoroalkylalkoxy, dialkylamino, or aryloxy. 
Carboxymethoxylamine hemihydrochloride (1) was esterified with the appropriate alcohol saturated with hydrogen chloride. The reaction was carried out at ambient temperature for 24 hours. The hydrochloride salt of the ester (2) was obtained. Reaction of 2 with ethyl- or methyl-chloroformate in alcohol in the presence of base such as sodium bicarbonate afforded the oxy-carbamate (3). This reaction was carried out at ambient temperature over a period of 3-5 hours. Compound (3) was alkylated with alkyl iodides (excess) by refluxing in alcohol under basic conditions over a period of 2 to 3 hours affording 4. Hydrolysis of 4 to the N-alkyl-carboxymethoxylamine hydrochloride (5) was carried out by refluxing with hydrochloric acid over 20 minutes to one hour period. Esterification of 5 with the appropriate alcohol saturated with hydrogen chloride followed by basic workup afforded the N-alkyl-aminoxyacetate (6). Alternatively, ethyl aminoxy acetate (2a) was liberated from its hydrochloride salt with base, reaction of 2 with 2-5 fold excess of aldehydes or ketones either neat or in alcohol over a period of 1 to 4 hours under reflux afforded the imine (3a). Hydride reduction of (3a) under acidic condition in a solvent such as an alcohol followed by basic workup afforded the amine (6). Reaction of (6) with isothiocyanates in a solvent such as chloroform or ether over a period of 20 minutes to 1 hour provided compound (7).
The ability of the compounds of this invention to increase blood serum HDL levels was evaluated by the following standard experimental procedure for determination of HDL cholesterol:
Male Sprague-Dawley rats weighing 200-225 g were housed two per cage and fed Purina Rodent Chow Special Mix 5001-S supplemented with 0.25% cholic acid and 1.0% cholesterol and water ad libitum for 8 days. Each test substance was administered to a group of six rats fed the same diet with the test diet mixed in as 0.005-0.1% of the total diet. Body weight and food consumption were recorded prior to diet administration and at termination. Typical doses of the test substances are 5-100 mg/kg/day.
At termination, blood was collected from anesthetized rats and the serum was separated by centrifugation. Total serum cholesterol was assayed using the Sigma Diagnostics enzymatic kit for the determination of cholesterol, Procedure No. 352, modified for use with ninety-six well microtiter plates. After reconstitution with water the reagent contained 300 U/l cholesterol oxidase, 100 U/l cholesterol esterase, 1000 U/l horseradish peroxidase, 0.3 moles/l 4-aminoantipyrine and 30.0 moles/l p-hydroxybenzenesulfonate in a pH 6.5 buffer. In the reaction cholesterol is oxidized to produce hydrogen peroxide which is used to form a quinoneimine dye. The concentration of dye formed is measured spectrophotometrically by absorbence at 490 nm after incubation at 25xc2x0 C. for 30 minutes. The concentration of cholesterol was determined for each serum sample relative to a commercial standard from Sigma.
HDL cholesterol concentrations in serum were determined by separation of lipoprotein classes by fast protein liquid chromatography (FPLC) by a modification of the method of Kieft et al., J. Lipid Res., 32 (1991) 859-866. 25 ul of serum was injected onto Superose 12 and Superose 6 (Pharmacia), in series, with a column buffer of 0.05 M Tris (2-amino-2-hydroxymethyl-1,3-propanediol) and 0.15 M sodium chloride at a flow rate of 0.5 ml/min. The eluted sample was mixed on line with Boehringer-Mannheim cholesterol reagent pumped at 0.2 ml/min. The combined eluents were mixed and incubated on line through a knitted coil (Applied Biosciences) maintained at a temperature of 45xc2x0 C. The eluent was monitored by measuring absorbance at 490 nm and gave a continuous absorbance signal proportional to the cholesterol concentration. The relative concentration of each lipoprotein class was calculated as the per cent of total absorbency. HDL cholesterol concentration, in serum, was calculated as the per cent of total cholesterol as determined by FPLC multiplied by the total serum cholesterol concentration.
Test compounds were administered at a dose of 100 mg/kg. Each test substance was administered to a group of six rats. The duration of treatment was eight days. The compounds of the present invention were found to increase HDL cholesterol concentrations as summarized in Table I:
This invention also provides pharmaceutical compositions comprised amino thioxo methyl aminoxy acetic acid derivatives either alone or in combination with excipients (i.e. pharmaceutically acceptable materials with no pharmacological effects). Such compositions are useful in the treatment of atherosclerotic conditions such as dyslipoproteinemias and coronary heart disease, in that they increase the blood serum high density lipoprotein concentration of mammals treated with the compounds.
The precise dosage to be employed depends upon several factors including the host, whether in veterinary medicine or human medicine, the nature and severity of the condition being treated, the mode of administration and the particular active substance employed. The compounds may be administered by any conventional route, in particular enterally, preferably orally in the form of tablets or capsules. Administered compounds can be in the free form or pharmaceutically acceptable salt form as appropriate, for use as a pharmaceutical, particularly for use in the prophylactic or curative treatment of atherosclerosis and sequelae (angina pectoris, myocardial infarction, arrhythmias, heart failure, kidney failure stroke, peripheral arterial occlusion, and related disease states). These measures will slow the rate of progress of the disease state and assist the body in reversing the process direction in a natural manner.
Any suitable carrier known to the art can be used to prepare the pharmaceutical compositions. In such a composition, the carrier may be a solid, liquid or mixture of a solid and a liquid. Solid compositions include powders, tablets and capsules. A solid carrier can be one or more substances which may also act as a flavoring agent, lubricant, solubilizer, suspending agent, binder, or tablet disintegrant. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. Suitable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, hydroxymethyl cellulose, sodium carboxymethyl cellulose, a low melting wax, cocoa butter, and the like. Encapsulating materials may also be employed with the compounds of this invention, and the term xe2x80x9ccompositionxe2x80x9d is intended to include the active ingredient in combination with an encapsulating material as a formulation, with or without other carriers. Cachets may also be used in the delivery of the anti-atherosclerotic medicament of this invention.
Sterile liquid compositions include solutions, suspensions, emulsions, syrups and elixirs. The compounds of this invention may be dissolved or suspended in the pharmaceutically acceptable carrier, such as sterile water, sterile organic solvent or a mixture of both. Preferably the liquid carrier is one suitable for parental injection. Where the compounds are sufficiently soluble they can be dissolved directly in normal saline with or without the use of suitable organic solvents, such as propylene glycol or polyethylene glycol. If desired, dispersions of the finely divided compounds can be made-up in aqueous starch or sodium carboxymethyl cellulose solution, or in suitable oil, such as arachis oil. Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by intramuscular, intraperitoneal or subcutaneous injection. In many instances a liquid composition form may be used instead of the preferred solid oral method of administration.
It is preferred to prepare unit dosage forms of the compounds for standard administration regimens. In this way, the composition can be subdivided readily into smaller doses at the physicians direction. For example, unit dosages may be made up in packeted powders, vials or ampoules and preferably in capsule or tablet form. The active compound present in these unit dosage forms of the composition may be present in an amount of from about one gram to about fifteen grams or more, for single or multiple daily administration, according to the particular need of the patient. The daily dose of active compound will vary depending upon the route of administration, the size, age and sex of the patient, the severity of the disease state, and the response to the therapy as traced by blood analysis and the patient""s recovery rate. By initiating the treatment regimen with a minimal daily dose of about one gram, the blood levels of HDL and the patients symptomatic relief analysis may be used to determine whether a larger dose is indicated. Based upon the data presented below, the projected daily dose for both human and veterinary use will be from about 25 to about 200 milligrams/kilogram per day, and more usually, from about 50 to about 100 milligrams/kilogram per day.